Optical interrogation systems that utilize evanescent field-based sensors are fast becoming a technology of choice for accurate label-free detection of a biological, biochemical, or chemical substance (e.g., cells, spores, biological or drug molecules, or chemical compounds). This technology typically involves the use of grating-coupled waveguides (GCWs) to sense a concentration change, surface adsorption, reaction, or the mere presence of a biological or chemical substance at the waveguide surface. Evanescent field-based sensors typically utilize a planar grating-coupled waveguide in direct contact with the compounds that are immobilized on the surface of the waveguide. When a biological, bio-chemical or chemical reaction happens at the surface of the waveguide, it changes the refractive index over a thin layer (few nanometers thick) and, as a consequence, the effective refractive index of the grating-coupled waveguide also changes. When a light beam is sent to the grating-coupled waveguide, the light couples into the GCW (under resonant condition) and then reflects at one specific angle and at one specific wavelength. (These angles and wavelengths are referred to as resonant angles and resonant wavelengths.) These angles and wavelengths are a function of the waveguide's effective refractive index.
More specifically, the resonant condition occurs only for a specific wavelength and angle of the incoming light beam, and changes in either angle or wavelength of reflected (resonant) light correspond to changes in the effective refractive index of the GCW. Thus, the optical interrogation system is used to sense a change in the effective index of the GCW, which enables one to determine whether or not a substance of interest is located within the sensing region of the GCW. Therefore, by measuring the resonant angles and/or the wavelengths of the reflected light, one can detect variation in the waveguide's effective refractive index and, thereby, detect the presence of biological, biochemical, or chemical substances.
In order for this technology to be viable, one must be able to accurately monitor the resonant angle(s) and/or the resonant wavelength(s). Optical interrogation systems utilizing evanescent field-based sensors employ two different interrogation methods. The first method is called the wavelength interrogation approach. It utilizes a collimated wide spectral band light beam incident at one at one specific angle at the GCW and uses a spectrometer to measure the (resonant) wavelength of the reflected light beam. The second method is called the angular interrogation approach. This method utilizes a single wavelength light beam, at multiple incidence angles to interrogate GCW. When utilizing this approach, the detector measures the (resonant) angle of the reflected light beam. The optical interrogation system that utilizes the angular interrogation approach is disclosed, for example, in U.S. Pat. No. 6,218,194 and is incorporated by reference herein.
However, in addition to reflecting light at the resonance wavelength or the resonance angle, grating-coupled waveguides (GCWs) also reflect light at other wavelengths and angles (parasitic geometric reflections), thus reducing the quality of the detected resonant signal. More specifically, because of these parasitic reflections one may see a low contrast weak resonance signal superimposed to a background of reflected light happening at all angles or wavelengths. The background lowers signal to noise ratio and makes the angular or wavelength measurement of the resonance less accurate. A second problem is caused by the reflections from the first (front) surface of the substrate or microplate, which are referred to herein as parasitic Fresnel reflections. Parasitic Fresnel reflections interfere with the resonance measurements and generate fringes in the angular or spectral space making the resonance detection very noisy and, due to temperature fluctuations and other factors, instable over time.
One example of wavelength interrogation system is shown on FIG. 1. A lens 108 collimates the light beam provided by the input fiber 106 which is connected to a broad spectral range light source 100. The collimated beam is then directed, at a specific angle, toward the sensor that includes the microplate 102 and the GCW 104. The reflected (resonant) light beam is collected by the lens 108 and imaged over the output fiber 107 which is connected to a spectrometer 101.
FIG. 2 shows an example of an angular interrogation system were the input fiber 106 is connected to a monochromatic light source 110 and the light beam is focused on GCW 104. After the light beams couples in GCW it is reflected at angles corresponding to the resonance condition and is imaged over a position sensitive detector 111.
However, as stated above, both of these approaches suffer from parasitic reflections which obscure the true resonance signal. To illustrate the problem, the curve 130 of FIG. 3 shows the simulated spectral resonance shape of the light reflected by the sensor. This curve is highly non symmetric and has a relatively bad contrast definition that is due only to the geometric reflections of the GCW across different wavelengths.
The problem of the parasitic Fresnel reflections happening at the first face of the microplate is illustrated by the curve 132 of FIG. 4. This resonance curve 132 is modulated at high frequency because of interference effects between the resonant light and the parasitic Fresnel reflections of the first face of the microplate 102. The phase and the frequency of this modulation is a function of the thickness of the microplate 102.
Some solutions for avoiding parasitic reflections, such as increasing the spatial and/or angular separation of the incident and reflected beams, are proposed in the literature (e.g., Cottier, K., et al., “Label-free Highly Sensitive Detection of (Small) Molecules by Wavelength Interrogation of Integrated Optical Chips,” Sensors and Actuators B: Chemical, June 2003). The principle of this approach lies in defining the reader optics in such a way that the light is injected into one area of the grating-coupled waveguide and is collected from another area of the grating-coupled waveguide. Such a configuration is illustrated on FIG. 5. Hence, the detector is not collecting the parasitic reflections that occur at the input side of GCW, but rather collects only the light that has propagated into the grating coupled waveguide.
The solution described by Cottier, K., et al., however, is limited to unidirectional light propagation. It is important, in some cases, to collect the light that is propagating within the GCW in both directions. Therefore, Cottier's solution is not compatible with a double resonance approach, such as the one described, for example, in U.S. patent application Ser. No. 10/676,352, filed Sep. 30, 2003, by Gollier et al, which is incorporated by reference herein. Another limitation of the Cottier's technique is that it can only be utilized when the light propagation distances within the waveguide are larger than the input light beam diameter. Furthermore, in some cases, we want to measure different propagation modes (TE and TM). These modes have significantly different propagation distances. It is impossible, in utilizing the Cottier approach, to find a distance between the incident input beam (on GCW) and the collected output beam (from GCW) that allows us to keep all the modes and to simultaneously filter out parasitic reflections. Finally, in some other cases, it may be desirable to sending the light beam at normal incidence with respect to the GCW. However, in this case, a stationary wave is excited within the GCW. Thus, using the spatial separation suggested by the Cottier is not possible, because the light is not propagating within the GCW.
To illustrate those problems, the FIG. 6a shows a 4 resonances spectrum produced by the device of FIG. 1. The two peaks 140 on the left side of the spectrum curve represent the two TM modes propagating in opposite directions. The two peaks 141 on the right side of the spectrum curve represent the two TE modes propagating in the two directions. FIG. 6a illustrates that peaks 140 are difficult to detect due to parasitic reflections and that all peaks suffer from high frequency modulation. When the input and output light beams are separated as taught by Cottier, so as to filter out parasitic reflections around one of the resonant wavelengths corresponding to one of TM modes, the resonant wavelength corresponding to the second TM mode can no longer be detected. Furthermore, the two TE modes (which have smaller propagation distances within the waveguide) begin to disappear into the background noise (due to parasitic reflections) and are now difficult to detect. This is illustrated in FIG. 6b. Thus, Cottier method can not be utilized if one needs to detect both TM and TE (resonant modes), and/or if one needs to detect resonant modes propagating in opposing directions.